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Desmoplakin (DP) is an obligate component of desmosomal cell-cell junctions that links the adhesion plaque to the cytoskeletal intermediate filament network. While a central role for DP in maintaining the structure and stability of the desmosome is well established, recent work has indicated that DP's functions may extend beyond cell-cell adhesion. In our study, we show that loss of DP results in a significant increase in cellular migration, as measured by scratch wound assays, Transwell migration assays, and invasion assays. Loss of DP causes dramatic changes in actin cytoskeleton morphology, including enhanced protrusiveness, and an increase in filopodia length and number. Interestingly, these changes are also observed in single cells, indicating that control of actin morphology is a cell-cell adhesion-independent function of DP. An investigation of cellular signaling pathways uncovered aberrant Rac and p38 mitogen-activated protein kinase (MAPK) activity in DP knockdown cells, restoration of which is sufficient to rescue DP-dependent changes in both cell migration and actin cytoskeleton morphology. Taken together, these data highlight a previously uncharacterized role for the desmosomal cytolinker DP in coordinating cellular migration via p38 MAPK and Rac signaling.

Desmosomal mutations result in potentially deadly cardiocutaneous disease caused by electrical conduction defects and disruption of gap junctions. Kam et al. demonstrate a mechanism whereby loss of the intermediate filament anchoring protein desmoplakin stimulates Cx43 turnover by increasing K-Ras expression, marking Cx43 for lysosomal degradation through ERK1/2 phosphorylation., Desmoplakin (DP) is an obligate component of desmosomes, intercellular adhesive junctions that maintain the integrity of the epidermis and myocardium. Mutations in DP can cause cardiac and cutaneous disease, including arrhythmogenic cardiomyopathy (ACM), an inherited disorder that frequently results in deadly arrhythmias. Conduction defects in ACM are linked to the remodeling and functional interference with Cx43-based gap junctions that electrically and chemically couple cells. How DP loss impairs gap junctions is poorly understood. We show that DP prevents lysosomal-mediated degradation of Cx43. DP loss triggered robust activation of ERK1/2–MAPK and increased phosphorylation of S279/282 of Cx43, which signals clathrin-mediated internalization and subsequent lysosomal degradation of Cx43. RNA sequencing revealed Ras-GTPases as candidates for the aberrant activation of ERK1/2 upon loss of DP. Using a novel Ras inhibitor, Ras/Rap1-specific peptidase (RRSP), or K-Ras knockdown, we demonstrate restoration of Cx43 in DP-deficient cardiomyocytes. Collectively, our results reveal a novel mechanism for the regulation of the Cx43 life cycle by DP in cardiocutaneous models.

The GEF Bcr promotes RhoA-dependent actin remodeling and MAL/SRF signaling in keratinocytes, which in turn promotes differentiation via regulation of desmoglein-1 expression., Although much is known about signaling factors downstream of Rho GTPases that contribute to epidermal differentiation, little is known about which upstream regulatory proteins (guanine nucleotide exchange factors [GEFs] or GTPase-activating proteins [GAPs]) are involved in coordinating Rho signaling in keratinocytes. Here we identify the GEF breakpoint cluster region (Bcr) as a major upstream regulator of RhoA activity, stress fibers, and focal adhesion formation in keratinocytes. Loss of Bcr reduced expression of multiple markers of differentiation (such as desmoglein-1 [Dsg1], keratin-1, and loricrin) and abrogated MAL/SRF signaling in differentiating keratinocytes. We further demonstrated that loss of Bcr or MAL reduced levels of Dsg1 mRNA in keratinocytes, and ectopic expression of Dsg1 rescued defects in differentiation seen upon loss of Bcr or MAL signaling. Taken together, these data identify the GEF Bcr as a regulator of RhoA/MAL signaling in keratinocytes, which in turn promotes differentiation through the desmosomal cadherin Dsg1.

We have recently shown that a fraction of the total cellular pool of the small GTPase RhoA resides in the nucleus, and that the nuclear guanine nucleotide exchange factor (GEF) Net1 has a role in the regulation of its activity. In this protocol, we describe a method to measure both the activities of the nuclear pools of RhoA and Rho GEFs. This process required the development of a nuclear isolation protocol that is both fast and virtually free of cytosolic and membrane contaminants, as well as a redesign of existing RhoA and Rho GEF activity assays so that they work in nuclear samples. This protocol can be also used for other Rho GTPases and Rho GEFs, which have also been found in the nucleus. Completion of the procedure, including nuclear isolation and RhoA or Rho GEF activity assay, takes 1 h 40 min. We also include details of how to perform a basic assay of whole-cell extracts.

Objective— The goals of this study were to identify the signaling pathway by which sphingosine 1-phosphate (S1P) activates RhoA in smooth muscle cells (SMC) and to evaluate the contribution of this pathway to the regulation of SMC phenotype. Methods and Results— Using a combination of receptor-specific agonists and antagonists we identified S1P receptor 2 (S1PR2) as the major S1P receptor subtype that regulates SMC differentiation marker gene expression. Based on the known coupling properties of S1PR2 and our demonstration that overexpression of Gα 12 or Gα 13 increased SMC-specific promoter activity, we next tested whether the effects of S1P in SMC were mediated by the regulator of G protein-signaling-Rho guanine exchange factors (RGS-RhoGEFs) (leukemia-associated RhoGEF [LARG], PDZ-RhoGEF [PRG], RhoGEF [p115]). Although each of the RGS-RhoGEFs enhanced actin polymerization, myocardin-related transcription factor-A nuclear localization, and SMC-specific promoter activity when overexpressed in 10T1/2 cells, LARG exhibited the most robust effect and was the only RGS-RhoGEF activated by S1P in SMC. Importantly, siRNA-mediated depletion of LARG significantly inhibited the activation of RhoA and SMC differentiation marker gene expression by S1P. Knockdown of LARG had no effect on SMC proliferation but promoted SMC migration as measured by scratch wound and transwell assays. Conclusion— These data indicate that S1PR2-dependent activation of RhoA in SMC is mediated by LARG and that this signaling mechanism promotes the differentiated SMC phenotype.

The desmosomal armadillo protein plakophilin 2 (PKP2) regulates cell contact-initiated cortical actin remodeling through the regulation of RhoA localization and activity to couple adherens junction maturation with desmosomal plaque assembly., Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome. We previously showed that PKP2 loss prevents the incorporation of desmosome precursors enriched in the plaque protein desmoplakin (DP) into newly forming desmosomes, in part by disrupting PKC-dependent regulation of DP assembly competence. On the basis of the observation that DP incorporation into junctions is cytochalasin D–sensitive, here we ask whether PKP2 may also contribute to actin-dependent regulation of desmosome assembly. We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization. Our data suggest that these defects result from the failure of activated RhoA to localize at intercellular interfaces after cell–cell contact and an elevation of cellular RhoA, stress fibers, and other indicators of contractile signaling in squamous cell lines and atrial cardiomyocytes. Consistent with these observations, RhoA activation accelerated DP redistribution to desmosomes during the first hour of junction assembly, whereas sustained RhoA activity compromised desmosome plaque maturation. Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP–IF interactions required for normal desmosome assembly.

Despite decades of research, cancer remains one of the leading causes of death worldwide. Progression of cancer includes the breakdown or loss of normal tissue structure, which closely depends on the proper expression and regulation of numerous cell–cell adhesion molecules. Not surprisingly, the multifunctional cadherin cell–cell adhesion protein family members have emerged as critical regulators of tumorigenesis. The maintenance of cell–cell junctions and adhesion-mediated signaling pathways are tightly regulated by cadherin expression in a tissue-specific manner. In addition to their adhesive functions, cadherins integrate diverse cellular inputs (from cell–cell adhesion to mechanical forces or receptor tyrosine kinase activity) and translate these cues into biochemical intracellular signaling events involved in cell proliferation, motility, survival, and tissue homeostasis. Alterations in cadherin function can lead to cancer progression through a variety of molecular mechanisms including cadherin switching/EMT and the misregulation of different signaling mediators, including Rho GTPases, Ras/MAPK, Hippo/YAP, PI3K/Akt, and other pathways that have been implicated in tumor progression. Furthermore, cadherins have been recently implicated in mechanotransduction and cancer stem cell signaling. In this chapter, we report both fundamental findings and novel insights that define the roles of cadherins in human cancer and discuss how changes in the expression and regulation of these molecules contribute to cancer progression.

Net1 is a RhoA-specific guanine nucleotide exchange factor which localizes to the nucleus at steady state. A deletion in its N terminus redistributes the protein to the cytosol, where it activates RhoA and can promote transformation. Net1 contains a PDZ-binding motif at the C terminus which is essential for its transformation properties. Here, we found that Net1 interacts through its PDZ-binding motif with tumor suppressor proteins of the Dlg family, including Dlg1/SAP97, SAP102, and PSD95. The interaction between Net1 and its PDZ partners promotes the translocation of the PDZ proteins to nuclear subdomains associated with PML bodies. Interestingly, the oncogenic mutant of Net1 is unable to shuttle the PDZ proteins to the nucleus, although these proteins still associate as clusters in the cytosol. Our results suggest that the ability of oncogenic Net1 to transform cells may be in part related to its ability to sequester tumor suppressor proteins like Dlg1 in the cytosol, thereby interfering with their normal cellular function. In agreement with this, the transformation potential of oncogenic Net1 is reduced when it is coexpressed with Dlg1 or SAP102. Together, our results suggest that the interaction between Net1 and Dlg1 may contribute to the mechanism of Net1-mediated transformation.

The process of fibrosis, described as accumulation of myofibroblasts and excessive deposition of extracellular matrix components, is a key development in the progression of multiple different types of cardiac disease. Nevertheless, little is known about the molecular mechanisms which cause the onset of fibrosis in cardiac disease. Fibrosis is a significant component of arrhythmogenic cardiomyopathy (AC), a genetic disorder characterized by replacement of healthy cardiomyocytes (CMs) with fibrous tissue, leading to arrhythmia and in certain cases, sudden death. AC is often characterized as a “disease of the desmosome”, as mutations for all obligate desmosome proteins have been found in cases of AC, including the desmosome armadillo proteins Plakophilin-2 (PKP2) and Plakoglobin (PG). PKP2 and PG are multi-functional proteins involved in both mechanical stabilization of the cardiac area composita, as well as mediation of desmosome-related signaling pathways. We have determined that loss of PKP2 or PG in neonatal CMs causes an aberrant increase in gene expression of pro-fibrotic stimuli such as transforming growth factor beta 1 (TGF-beta1) and Interleukin-6 (IL-6). In addition, p38 MAPK, a known mediator of inflammatory fibrosis, is activated upon loss of PKP2/PG. We hypothesize that mutation or loss of PKP2 or PG cause the recruitment and activation of cardiac fibroblasts via pro-fibrotic TGF-beta and p38MAPK signaling, resulting in pathological fibrosis characteristic of AC. Indeed, conditioned media from PKP2-silenced CMs causes an increase in fibronectin gene expression by freshly isolated cardiac fibroblasts. Our future experiments will investigate whether inhibition of TGF-beta or p38MAPK signaling can alleviate fibrotic gene production. By highlighting a novel link between desmosome armadillo proteins and pro-fibrotic signaling in cardiac tissue, this study provides mechanistic insights into the pathogenesis of AC, as well as advances our knowledge of potential therapeutic targets for combating fibrosis in multiple different types of heart disease or injury.

Genetic disorders of the Ras/MAPK pathway, termed RASopathies, produce numerous abnormalities, including cutaneous keratodermas. The desmosomal cadherin, desmoglein-1 (DSG1), promotes keratinocyte differentiation by attenuating MAPK/ERK signaling and is linked to striate palmoplantar keratoderma (SPPK). This raises the possibility that cutaneous defects associated with SPPK and RASopathies share certain molecular faults. To identify intermediates responsible for executing the inhibition of ERK by DSG1, we conducted a yeast 2-hybrid screen. The screen revealed that Erbin (also known as ERBB2IP), a known ERK regulator, binds DSG1. Erbin silencing disrupted keratinocyte differentiation in culture, mimicking aspects of DSG1 deficiency. Furthermore, ERK inhibition and the induction of differentiation markers by DSG1 required both Erbin and DSG1 domains that participate in binding Erbin. Erbin blocks ERK signaling by interacting with and disrupting Ras-Raf scaffolds mediated by SHOC2, a protein genetically linked to the RASopathy, Noonan-like syndrome with loose anagen hair (NS/LAH). DSG1 overexpression enhanced this inhibitory function, increasing Erbin-SHOC2 interactions and decreasing Ras-SHOC2 interactions. Conversely, analysis of epidermis from DSG1-deficient patients with SPPK demonstrated increased Ras-SHOC2 colocalization and decreased Erbin-SHOC2 colocalization, offering a possible explanation for the observed epidermal defects. These findings suggest a mechanism by which DSG1 and Erbin cooperate to repress MAPK signaling and promote keratinocyte differentiation.

Loss of the desmosome armadillo protein Plakophilin-2 in neonatal cardiomyocytes results in decreased stability and expression of the cytoskeletal linker protein Desmoplakin, which causes activation of a TGF-β1/p38 MAPK signaling cascade and induces expression of fibrotic genes., Members of the desmosome protein family are integral components of the cardiac area composita, a mixed junctional complex responsible for electromechanical coupling between cardiomyocytes. In this study, we provide evidence that loss of the desmosomal armadillo protein Plakophilin-2 (PKP2) in cardiomyocytes elevates transforming growth factor β1 (TGF-β1) and p38 mitogen-activated protein kinase (MAPK) signaling, which together coordinate a transcriptional program that results in increased expression of profibrotic genes. Importantly, we demonstrate that expression of Desmoplakin (DP) is lost upon PKP2 knockdown and that restoration of DP expression rescues the activation of this TGF-β1/p38 MAPK transcriptional cascade. Tissues from PKP2 heterozygous and DP conditional knockout mouse models also exhibit elevated TGF-β1/p38 MAPK signaling and induction of fibrotic gene expression in vivo. These data therefore identify PKP2 and DP as central players in coordination of desmosome-dependent TGF-β1/p38 MAPK signaling in cardiomyocytes, pathways known to play a role in different types of cardiac disease, such as arrhythmogenic or hypertrophic cardiomyopathy.

We previously showed that the cell–cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here we show that PG deficiency results in extracellular matrix (ECM)-dependent disruption of mature focal adhesions and cortical actin organization. Plating PG−/− cells onto ECM deposited by PG+/− cells partially restored normal cell morphology and inhibited PG−/− cell motility. In over 70 adhesion molecules whose expression we previously showed to be altered in PG−/− cells, a substantial decrease in fibronectin (FN) in PG−/− cells stood out. Re-introduction of PG into PG−/− cells restored FN expression, and keratinocyte motility was reversed by plating PG−/− cells onto FN. Somewhat surprisingly, based on previously reported roles for PG in regulating gene transcription, PG-null cells exhibited an increase, not a decrease, in FN promoter activity. Instead, PG was required for maintenance of FN mRNA stability. PG−/− cells exhibited an increase in activated Src, one of the kinases controlled by FN, a phenotype reversed by plating PG−/− cells on ECM deposited by PG+/− keratinocytes. PG−/− cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PG−/− keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM–Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability.

Focal adhesions have been intensely studied ever since their discovery in 1971. The last three decades have seen major advances in understanding the structure of focal adhesions and the functions they serve in cellular adhesion, migration, and other biological processes. In this chapter, we begin with a historical perspective of focal adhesions, provide an overview of focal adhesion biology, and highlight recent major advances in the field. Specifically, we review the different types of matrix adhesions and the role different Rho GTPases play in their formation. We discuss the relative contributions of integrin and syndecan adhesion receptors to the formation of focal adhesions. We also focus on new insights gained from studying focal adhesions on biomaterial surfaces and from the growing field of mechanotransduction. Throughout this chapter, we have highlighted areas of focal adhesion biology where major questions still remain to be answered.

Rho guanine nucleotide dissociation inhibitors (RhoGDIs) bind to inactive Rho GTPases in the cytosol, but their function remains unclear. Several Rho GTPases are now shown to compete for RhoGDI binding and this is crucial for regulating Rho GTPase turnover and activation. At steady state, most Rho GTPases are bound in the cytosol to Rho guanine nucleotide dissociation inhibitors (RhoGDIs)1. RhoGDIs have generally been considered to hold Rho proteins passively in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while activating the remaining membrane-bound fraction. Because RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression itself but from additional effects on the levels and activity of other Rho GTPases as a result of competition for binding to RhoGDI1; this may require a re-evaluation of previously published studies that rely exclusively on these techniques.

Focal adhesions have been intensely studied ever since their discovery in 1971. The last three decades have seen major advances in understanding the structure of focal adhesions and the functions they serve in cellular adhesion, migration, and other biological processes. In this chapter, we begin with a historical perspective of focal adhesions, provide an overview of focal adhesion biology, and highlight recent major advances in the field. Specifically, we review the different types of matrix adhesions and the role different Rho GTPases play in their formation. We discuss the relative contributions of integrin and syndecan adhesion receptors to the formation of focal adhesions. We also focus on new insights gained from studying focal adhesions on biomaterial surfaces and from the growing field of mechanotransduction. Throughout this chapter, we have highlighted areas of focal adhesion biology where major questions still remain to be answered.

Adhesion of cells to extracellular matrix proteins such as fibronectin initiates signaling cascades that affect cell morphology, migration and survival. Some of these signaling pathways involve the Rho family of GTPases, such as Cdc42, Rac1 and RhoA, which play a key role in regulating the organization of the cytoskeleton. Although significant advances have been made in understanding how Rho proteins control cytoskeletal architecture, less is known about the signals controlling activation of the GTPases themselves. The focus of this study was to determine which guanine nucleotide exchange factor(s) are responsible for activation of RhoA downstream of adhesion to fibronectin. Using an affinity pulldown assay for activated exchange factors, we show that the RhoA-specific exchange factors Lsc/p115 RhoGEF and LARG are activated when cells are plated onto fibronectin, but not other exchange factors such as Ect2 or Dbl. Knockdown of Lsc and LARG together significantly decreases RhoA activation and formation of stress fibers and focal adhesions downstream of fibronectin adhesion. Similarly, overexpression of a catalytically inactive mutant of Lsc/p115 RhoGEF inhibits RhoA activity and formation of stress fibers and focal adhesions on fibronectin. These data establish a previously uncharacterized role for the exchange factors Lsc/p115 RhoGEF and LARG in linking fibronectin signals to downstream RhoA activation.